Polyarylates for drug delivery and tissue engineering

ABSTRACT

Biocompatible polyarylates of tyrosine-derived diphenol compounds and poly(alkylene oxide) dicarboxylic acids, articles formed therefrom and therapeutic uses are disclosed.

FIELD OF THE INVENTION

The present invention relates to bioerodable polyarylates co-polymers oftyrosine-derived dipeptides and functionalized poly(alkylene oxides).The present invention further relates to injectable composition andimplantable articles for drug delivery and tissue engineering made fromthese polymers.

BACKGROUND

The biotechnology industry has evolved to allow the large scaleproduction of recombinant proteins for commercial use. However, many ofthese therapeutic proteins and vaccines present a unique challenge fordrug delivery. The successful clinical application or commercial use ofmany proteins requires the use of a protein delivery system to reducethe frequency of administration, provide a lower toxicity through areduced peak serum concentration, localize the drug at the site ofaction, or yield a steady-state level of the drug to achieve the desiredeffect. The biggest challenge however is to maintain the stability ofproteins during the formulation steps.

Proteins are generally not very stable, as the stabilization energy ofthe native state is mostly between 5 and 20 kcal/mol, which isequivalent to that of a few hydrogen bonds. Many forces are involved inkeeping the native proteins properly folded including hydrophobicinteractions, electrostatic interactions (charge repulsion and ionpairing), hydrogen bonding, intrinsic propensities, and van der Waalsforces. Among these forces, hydrophobic interactions seem to be thedominant.

There are many factors that affect protein stability. These include atleast temperature, pH, ionic strength, metal ions, surface adsorption,shearing, shaking, additives, solvents, protein concentration, purity,morphism, pressure, freeze-thawing and drying. Most mesophilic proteins,such as those from human beings, can be denatured easily at temperaturesbetween 50 to 80° C. A common phenomenon of protein instability is theformation of protein aggregates, which can be soluble or insoluble,chemical or physical, and reversible or irreversible. Chemicaltransformations that lead to protein instability include at leastdeamidation, oxidation, hydrolysis, isomerization, succinimidation,disulfide bond formation and breakage, non-disulfide cross linking anddeglycosilation. It is therefore clear that developing a delivery systemfor proteins is not a trivial matter.

The most common approach used to develop a controlled delivery systemfor proteins is to encapsulate the protein in a polymer matrix ormicrosphere of poly(lactic-co-glycolic acid) (PLGA), which has been usedfor over twenty years as a resorbable suture material. A great deal ofresearch has been published on the development of protein-controlledrelease formulations using PLGA microspheres. However, there are only asmall number of successful commercial formulations using this system. Abiodegradable microsphere formulation, the Lupron Depot, has beencommercially successful for several years. This formulation consists ofleuprolide acetate, a decapeptide, encapsulated in biodegradablemicrospheres of PLGA for the treatment of prostate cancer,endometriosis, and precocious puberty. The drug is released continuouslyover either one or three months depending upon the formulation. Abiodegradable human growth hormone microsphere formulation is presentlymarketed by Alkermes, Inc.

PLGA microspheres are usually prepared by a solvent-based process, whichas discussed above, is harmful to protein stability and activity. Meltprocessing is not a viable alternative, because the T_(g) of the PLGAsystem is in the range of 55 to 60° C. Thus, melt processing has to beconducted at relatively high temperatures that are detrimental to thestability and activity of the proteins. PLGA degradation also releasesacidic byproducts, which can inactivate the proteins.

Drug delivery systems require a product that is injectable through a 21gauge needle and from which a protein can be delivered at a sustainedrate for two to four weeks without loss of activity. Water may be usedas a diluent to allow injection through a narrow bore needle, andstabilizers such as sugars or salts may be used.

Accordingly, there exists a need for a biocompatible low viscositypolymer that is degradable and resorbable and able to absorb water or beemulsified in water so that water can be used as a diluent, ifnecessary. Ideally, for drug delivery purposes, the polymer should befluid at room temperature. However, polymers that become fluid withheating to temperature below the temperature at which proteins denatureare also suitable.

SUMMARY OF THE INVENTION

This need is met by the present invention. It has now been discoveredthat tyrosine-derived diphenols can be copolymerized with poly(alkyleneoxide) dicarboxylic acids to form non-toxic bioerodable polyarylatesmeeting the above drug delivery system requirements. Higher molecularweight and viscosity versions of these polymers also possess desirabletissue engineering properties.

Therefore, according to one aspect of the present invention,polyarylates are provided that have repeating structural units accordingto Formula I

wherein R₁ is CH═CH or (CH₂)_(n) wherein n is from 0 to 18, inclusive;R₂ is selected from hydrogen and straight and branched alkyl andalkylaryl groups containing up to 18 carbon atoms; and R has thestructure of Formula IIa or IIb:—(CH₂—)_(a O—[(CH) ₂—)_(a)CHR₃—O—]_(m)(CH₂—)_(a)  (IIa)and—R₄—C(═O)—O[(—CH₂)_(a)—CHR₃—O—]_(m)C(═O)—R₄—  (IIb)wherein a is from 1 to 3, inclusive, m is from 1 to 500,000, inclusive,R₃ is hydrogen or a lower alkyl group containing from one to four carbonatoms, and R₄ is selected from a bond or straight and branched alkyl andalkylaryl groups containing up to 18 carbon atoms.

The pendant side chains on each polyarylate repeating unit provide afurther degree of freedom in the design of the polyarylates and can beused to modify the overall physicomechanical properties of the polymerwithout changing the polymer backbone structure. This allows for thesystemic variation in polymer structure, which leads to the developmentof structure-property relationships. The polyarylates of the presentinvention are expected to exhibit orthogonal relationships, which allowsthe variation of many properties independent of others.

The polymers of the present invention have low melting/softening points,which will facilitate their formulation with temperature sensitivebiologically or pharmaceutically active compounds such as proteins bypermitting the active compounds and polymers to be directly mixedwithout solvents or heating. Therefore, according to another embodimentof the present invention, the polymers are combined with a quantity ofbiologically or pharmaceutically active compound sufficient foreffective site-specific or systemic drug delivery as described byGutowska, et al., J. Biomater. Res., 29, 811-21 (1995), and Hoffman, J.Controlled Release, 6, 297-305 (1987). The biologically orpharmaceutically active compound may be physically admixed, embedded inor dispersed in the polymer matrix.

Accordingly, in yet another aspect of the present invention, a method isprovided for site-specific or systemic drug delivery by administering toa patient in need thereof a therapeutically effective amount of abiologically or pharmaceutically active compound in combination with thepolymer of the present invention. The polymer drug combinations of thepresent invention include combinations in which the polymer functions asa degradable matrix for the suspension of drug loaded microparticles tocontrol the rate of release and to prevent microparticle migration.

Implantable medical materials and devices formed from the polymers ofthe present invention are also included in the scope of the presentinvention. In accordance with this aspect of the present invention, thepolymers of the present invention may be used as binding agents formaking shapable putties from solid materials such as hydroxyapatite,calcium sulfate, tricalcium phosphate, demineralized bone matrix,bioglass, and the like, or used alone as a temporary filling material incosmetic reconstructive surgery.

The poly(alkylene oxide)monomeric repeating units decrease the surfaceadhesion of the polymers of the present invention. As the value of m inFormulae IIa and IIb increases, the surface adhesion decreases. Polymercoatings may thus be prepared that are resistant to cell attachment anduseful as non-thrombogenic coatings on surfaces in contact with blood.Such polymers also resist bacterial adhesion in this, and in othermedical applications as well. The present application therefore includesblood contacting devices and medical implants having surfaces coatedwith the polymers of the present invention. The surfaces are preferablypolymeric surfaces. The methods according to the present inventioninclude implanting in the body of a patient a blood-contacting device ormedical implant having a surface coated with the polymers of the presentinvention.

The polymers of the present invention have good film-forming properties.An important phenomenon observed is the temperature-dependent inversephase transition of the polymers in aqueous solution. As temperatureincreases, the polymers undergo an inverse phase transition to acollapsed state. Stated another way, the polymers form gels uponheating. Therefore, the present invention also includes aqueous polymercompositions that gel upon heating. Polymers that undergo the inversephase transition at about 30 to 40° C. on heating can be used asbiomaterials for drug release and clinical implantation materials.Specific applications include films and sheets for the prevention ofadhesion and for tissue reconstruction.

Therefore, in another embodiment of the present invention, the polymersof the present invention may be formed into a sheet or a coating forapplication to exposed injured tissues for use as a barrier for theprevention of surgical adhesions as described by Urry, et al., Mat. Res.Soc. Symp. Proc., 292, 253-264 (1993). Therefore, another aspect of thepresent invention provides a method for preventing the formation ofadhesion between injured tissues by inserting as a barrier between theinjured tissues a sheet or coating of the polymers of the presentinvention.

A more complete appreciation of the invention and many other intendedadvantages can be readily obtained by reference to the followingDetailed Description of the Preferred Embodiment and claims, whichdisclose the principles of the invention and the best modes which arepresently contemplated for carrying them out.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the release of BSA from DTR-PEG 600;

FIG. 2 depicts BSA Release from DTR-PEG-1000 Succinates (LMW);

FIG. 3 depicts the effect of PEG molecular weight on BSA release fromDTR-PEG 1000-Succinate;

FIG. 4 depicts the effect of polymer molecular weight on BSA releasefrom DTR-PEG 1000-Succinate; and

FIG. 5 depicts the effect of poly(alkylene oxide) structure on BSArelease from DTR-PEG 1000 Succinate polymers.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The polyarylates of the present invention are prepared by thecondensation of a diacid with a diphenol according to the methoddescribed by U.S. Pat. No. 5,216,115 in which diphenol compounds arereacted with aliphatic or aromatic dicarboxylic acids in a carbodiimidemediated direct polyesterification using4-(dimethylamino)-pyridinium-p-toluene sulfonate (DPTS) as a catalyst.The disclosure of U.S. Pat. No. 5,216,115 in this regard is incorporatedherein by reference.

The diphenol compounds are the tyrosine-derived diphenol monomers ofU.S. Pat. Nos. 5,587,507 and 5,670,602, the disclosures of both of whichare also incorporated herein by reference. The polyarylates of Formula Iare prepared using tyrosine-derived diphenol monomers having thestructure of Formula III:

wherein R₁, R₂ and n are the same as described above with respect toFormula I.

The preferred diphenol monomers are desaminotyrosyl-tyrosine carboxylicacids and esters thereof, wherein R₁ is —CH₂—CH₂—, which are referred toas DT esters. For purposes of the present invention, the ethyl ester(R₂=ethyl) is referred to as DTE, the benzyl ester (R₂=benzyl) as DTBn,and so forth. Both patents disclose methods by which these monomers maybe prepared. For purposes of the present invention, thedesaminotyrosyl-tyrosine free carboxylic acid (R₂=hydrogen) is referredto as DT.

It is not possible to polymerize the polyarylates having pendant freecarboxylic acid groups from corresponding diphenols with pendant freecarboxylic acid groups without cross-reaction of the free carboxylicacid groups with the co-monomer. Accordingly, polyarylates that arehomopolymers or copolymers of benzyl ester diphenyl monomers such asDTBn may be converted to corresponding free carboxylic acid homopolymersand copolymers through the selective removal of the benzyl groups by thepalladium catalyzed hydrogenolysis method disclosed by co-pending andcommonly owned U.S. Pat. No. 6,120,491. The disclosure of this patent isincorporated by reference. The catalytic hydrogenolysis is necessarybecause the lability of the polymer backbone prevents the employment ofharsher hydrolysis techniques.

The dicarboxylic acids are derived from poly(alkylene oxides) such aspolyethylene glycol, polypropylene glycol, polybutylene glycol,Pluronics and the like. Polyethylene glycol diacids are preferred.

Two classes of diacids are disclosed. The first class produces polymersin which R has a structure according to Formula IIa. These diacids havethe structure of Formula IVa:HOOC—(CH₂—)_(a)O—[(CH₂—)_(a)CHR₃—O—]_(m)(CH₂—)_(a)COOH  (IVa)wherein a, m and R₃ are the same as described above with respect toFormula IIa. R₃ is preferably hydrogen, a is preferably 1, and m ispreferably between about 10 and about 100, and more preferably betweenabout 10 and about 50.

The diacids of Formula IVa are formed by the oxidation of poly(alkyleneoxides) according to well-known methods. One example of such a compoundis biscarboxymethyl polyethylene glycol, which is commerciallyavailable.

The second class of diacids produces polymers in which R has a structureaccording to Formula IIb. These diacids have the structure of FormulaIVb:HOOC—R₄—C(═O)—O[(—CH₂)_(a)—CHR₃—O—]_(m)C(═O)—R₄—COOH  (IVb)wherein a, m and R₃ are again the same as described above with respectto Formulae IIa or IIb. Again, R₃ is preferably hydrogen, a ispreferably 1, and m is preferably between about 10 and about 100, andmore preferably between about 10 and about 50. R₄ is the same asdescribed above with respect to Formula IIb.

The dicarboxylic acids of Formula IVb are poly(alkyleneoxides)bis-functionalized with dicarboxylic acids having the structureof Formula V:HOOC—R₄—COOH  (V)wherein R₄ is the same as described above with respect to Formula IIB,and preferably contains up to 12 carbon atoms.

The poly(alkylene oxides) of Formula IVb that are bis-functionalizedwith dicarboxylic acid are prepared by the reaction of anon-functionalized poly(alkylene oxide) with an excess of either thedicarboxylic acid (mediated by a coupling agent such as dicyclohexylcarbodiimide), the anhydride (e.g. succinic anhydride) in the presenceof pyridine or triethylamine, or a dicarboxylic acid chloride (e.g.adipoyl chloride) in the presence of an acid acceptor liketriethylamine.

For the dicarboxylic acids of Formula IVa, R₄ is preferably selected sothat the dicarboxylic acids employed as starting materials tobis-functionalize the poly(alkylene oxides) are either importantnaturally-occurring metabolites or highly biocompatible compounds.Preferred Formula V dicarboxylic acid starting materials thereforeinclude the intermediate dicarboxylic acids of the cellular respirationpathway known as the Krebs cycle. These dicarboxylic acids includealpha-ketoglutaric acid, succinic acid, fumeric acid, malic acid andoxaloacetic acid, for which R₄ is —CH₂—CH₂—C(═O)—, —CH₂—CH₂—, —CH═CH═,—CH₂—CH(—OH)— and —CH₂—C(═O)—, respectively.

Another naturally-occurring, preferred dicarboxylic acid startingmaterial is adipic acid (R₄=(—CH₂—)₄), found in beet juice. Otherpreferred biocompatible dicarboxylic acids include oxalic acid (no R₄),malonic acid (R₄═—CH₂—), glutaric acid (R₄=(CH_(2 —)) ₃, pimellic acid(R₄=(—CH₂—)₅, suberic acid (R₄=(—CH₂—)₆ and azalaic acid (R₄=(—CH₂—)₇.In other words, among the dicarboxylic acids suitable for use in thepresent invention are compounds in which R₄ represents (—CH₂—)_(z)wherein z is an integer between 0 and 12, inclusive. A preferred classof highly biocompatible aromatic dicarboxylic acids are thebis(p-carboxyphenoxy) alkanes such as bis(p-carboxyphenoxy)propane.

The polyarylates of the present invention degrade by hydrolysis into theoriginal starting materials, i.e., the tyrosine-derived diphenols andthe poly(alkylene oxide) dicarboxylic acids. The poly(alkylene oxide)dicarboxylic acids that are poly(alkylene oxides)bis-functionalized withdicarboxylic acids further degrade to the starting poly(alkylene oxides)and dicarboxylic acids.

The polyarylates of the present invention are highly hydrophilic, whichis advantageous for polymeric drug delivery systems. However, thehydrophilic:hydrophobic balance of the polyarylates can be varied inseveral ways. The ester of the pendant chain of the diphenol can bechanged, with longer-chain ester groups increasing hydrophobicity.Increasing the molecular weight of the poly(alkylene oxide) orincreasing the number of carbons in the alkylene group of thepoly(alkylene oxide) will also increase hydrophobicity. Changing thedicarboxylic acid used to bis-functionalized the poly(alkylene oxide)will also change the hydrophilic:hydrophobic balance.

Preferred polyarylates have weight average molecular weights betweenabout 1,000 and 500,000 daltons, preferably between about 3,000 and50,000 daltons, and more preferably between about 5,000 and 15,000daltons. Molecular weights are calculated by gel permeationchromatography relative to polystyrene standards in tetrahydrofuranwithout further correction.

The molecular weights of the polyarylates can be controlled either bylimiting the reaction time or the ratios of either component. Molecularweights can also be controlled by the quantity of the carbodiimidecoupling reagent that is used. The viscosities of the polyarylates ofthe present invention can also be reduced by mixing with water to formeither an aqueous solution or emulsion of the polymer.

Iodine- and bromine-containing polymers are radio-opaque. These polymersand their methods of preparation are disclosed by U.S. Pat. No.6,475,577. The disclosure of this patent is incorporated herein byreference. Radio-opaque polymers include repeating structural units inwhich one or more hydrogens of an aromatic ring, an alkylene carbon, orboth, are replaced with an iodine or bromine atom. The polyarylates ofthe present invention may be similarly iodine- and bromine-substituted.Polyarylates according to the present invention comprising the repeatingstructural units of Formula I are radio-opaque when copolymerized withradio-opaque monomers so that the polyarylates also contain radio-opaquerepeating structural units, preferably one or more of the repeatingstructural units of Formula I in which one or more hydrogens of anaromatic ring, an alkylene carbon, or both, have been replaced with aniodine or bromine atom.

Cellular attachment, migration and proliferation on the surface of thepolyarylates can be modulated as a function of poly(alkylene oxide)content, with attachment, migration and proliferation decreasing aspoly(alkylene oxide) content increases. The present invention thereforeincludes methods for regulating cellular, attachment, proliferation andmigration on the surface of a polymeric substrate by contacting livingcells, tissues or biological fluids containing living cells with thepolyarylates of the present invention. The polyarylates of the presentinvention are therefore particularly well-suited for use as coatings onmedical implants, barriers for preventing the formation of adhesionsbetween injured tissues, and polymer scaffolds for tissue engineering.

The polyarylates of the present invention may be formed into porouspolymer scaffolds for tissue engineering by the methods disclosed byU.S. Pat. No. 6,103,255. The disclosure of this patent is alsoincorporated herein by reference. Porous polymer scaffolds prepared fromthe inventive polyarylates may incorporate an effective amount of thebiologically active substance that either promotes or prevents aparticular variety of cellular or tissue ingrowth. Examples of suchsubstances include cell attachment mediators (such as the peptidecontaining variations of the “RGD” integrilin binding sequence known toaffect cellular attachment), biologically active ligands, and substancesthat enhance or exclude particular varieties of cellular or tissueingrowth. Such substances include, for example, osteoinductivesubstances (such as bone morphogenic proteins (BMP)), epidermal growthfactor (EGF), fibroblast growth factor (FGF), platelet-derived growthfactor (PDGF), insulin-like growth factor (IGF-I and II), TGFβ, and thelike.

Any type of cell can be added to this scaffold for culturing andpossible implantation, including cells of the muscular and skeletalsystem (such as condrocytes, fibroblasts, muscle cells and osteocytes),parenchymal cells such as hepatocytes, pancreatic cells (including Isletcells), cells of intestinal origin, and other cells such as nerve cellsand skin cells, either as obtained from donors, from established cellculture lines, from embryonic and non-embryonic stem cells (human andnon-human), or even before or after genetic engineering.

The polyarylates can be worked up by known methods commonly known in thefield of synthetic polymers to produce a variety of useful articles withvaluable physical and chemical properties, all derived from tissuecompatible monomers. The useful articles can be shaped by conventionalpolymer-forming techniques. Molded articles prepared from thepolyarylates are useful as degradable biomaterials for medical implantapplication. Molded articles may be prepared from the polyarylates, orthe polyarylates may be coated on the surface of molded articles.Polymer coatings are particularly useful, because as poly(alkyleneoxide) content increases, protein deposition and tissue adhesiondecreases. Whether a portion of or the entire article, the polyarylatesdecompose harmlessly within a known period of time.

The polyarylates can also be formed into drug delivery systems thatdegrade to release pharmacologically or biologically active agentswithin a predictable controlled release time. The polyarylates of thepresent invention having low molecular weights, and consequently lowviscosities and melting/softening points (including polymers that areliquid at temperatures below protein denaturation temperatures and aslow as room temperature) are particularly well suited as carriers forinjectable delivery systems for pharmacologically active biomoleculessuch as proteins, peptides, vaccines and genes, and the like, as well asother small pharmacologically active molecules. The low viscosities andmelting/softening points permit the direct mixing of the active moleculeand the polymer. The polyarylate release rate is determined by thehydrophilic:hydrophobic balance of the polymer. The more hydrophilicpolymer, the faster the rate of release. Polymer degradation rate willalso increase as hydrophilicity increases.

The low viscosity and softening/molecular weight polyarylates are alsouseful in the preparation of biodegradable liposomes and surfactants,and can be used as binding agents for making shapable putties fromsolids like hydroxyapatite, demineralized bone matrix and bioglass.These polyarylates can also be used in the preparation of degradablematrices for the suspension of drug loaded microparticle to control therate of release and to prevent microsphere migration. Polyarylates thatare solid at body temperature can also be used as temporary fillingmaterials in cosmetic reconstructive surgery. Certain of thepolyarylates that undergo inverse phase transitions upon heating may beapplied as a room temperature liquid that then solidifies upon heatingto body temperature.

The polyarylates of the present invention make possible the developmentof drug delivery systems having a number of advantages over the systemsthat are currently available or are being developed. Because thepolyarylates are low viscosity materials, biologically andpharmaceutically active agents can be mixed in at room temperaturewithout the addition of heat or organic solvents, representing asignificant advance in the ease of product formulation. The presentinvention therefore also includes injectable delivery systems forbiologically and pharmaceutically active compounds formed by directlymixing the active compound and polymer. The delivery system and itsmethod of preparation are particularly well suited for use with activecompounds such as pharmacologically active proteins, peptides, vaccinesand genes, and the like, as well as other small pharmacologically activemolecules, wherein the resulting mixtures can be injected without theuse of excipients.

However, the mixtures form stable combinations with excipients, shouldthe use of an excipient be desired. Both co-monomers, i.e., thetyrosine-derived diphenol and the poly(alkylene oxide) function tostabilize peptides and proteins. Release rates are easily controlled byadjustment of the hydrophilic:hydrophobic balance. Hydrophilic proteinstabilizers, such as trehalose, may be optionally added. Finally,because the polyarylates degrade rapidly, this allows multiplesequential injections, if necessary. Degradation rates are controlled byaltering the hydrophilic:hydrophobic balance.

The drug delivery systems of the present invention are suitable forapplications where localized drug delivery is desired, as well as insituations where systemic delivery is desired. Therapeutically effectivedosages may be determined by either in vivo or in vitro methods. Foreach particular compound of the present invention, individualdeterminations may be made to determine the optimal dosage required. Therange of therapeutically effective dosages will naturally be influencedby the route of administration, the therapeutic objectives, and thecondition of the patient. For the various suitable routes ofadministration, the absorption efficiency must be individuallydetermined for each drug by methods well known in pharmacology.Accordingly, it may be necessary for the therapist to titer the dosageand modify the route of administration as required to obtain the optimaltherapeutic effect. The determination of effective dosage levels, thatis, the dosage levels necessary to achieve the desired result, will bewithin the ambit of one skilled in the art. Typically, applications ofcompound are commenced at lower dosage levels, with dosage levels beingincreased until the desired effect is achieved. The release rate of thedrug from the formulations of this invention are also varied within theroutine skill in the art to determine an advantageous profile, dependingon the therapeutic conditions to be treated.

A typical dosage might range from about 0.001 mg/kg to about 1000 mg/kg,preferably from about 0.01 mg/kg to about 100 mg/kg, and more preferablyfrom about 0.10 mg/kg to about 20 mg/kg. Advantageously, the compoundsof this invention may be administered several times daily, and otherdosage regimens may also be useful.

The compositions may be administered subcutaneously, intramuscularly,colonically, rectally, nasally, orally or intraperitoneally, employing avariety of dosage forms such as suppositories, implanted pellets orsmall cylinders, aerosols, oral dosage formulations and topicalformulations, such as ointments, drops and transdermal patches.

Acceptable pharmaceutical carriers for therapeutic use are well known inthe pharmaceutical field, and are described, for example, in Remington'sPharmaceutical Science, Mac Publishing Co., (A. R. Gennaro edt. 1985).Such materials are non-toxic to recipients at the dosages andconcentrations employed, and include diluents, solubilizers, lubricants,suspending agents, encapsulating materials, solvents, thickeners,dispersants, buffers such as phosphate, citrate, acetate and otherorganic acid salts, anti-oxidants such as ascorbic acid, preservatives,low molecular weight (less than about 10 residues) peptides such aspolyarginine, proteins such as serum albumin, gelatin orimmunoglobulins, hydrophilic polymers such as poly(vinylpyrrolindinone),amino acids such as glycine, glutamic acid, aspartic acid or arginine,monosaccharides, disaccharides, and other carbohydrates includingcellulose or its derivatives, glucose, mannose or dextrines, chelatingagents such as EDTA, sugar alcohols such as mannitol or sorbitol,counter-ions such as sodium and/or non-ionic surfactants such as tween,pluronics or PEG.

The polymer-drug combinations of this invention may be prepared forstorage under conditions suitable for the preservation of drug activityas well as maintaining the integrity of the polymers, and are typicallysuitable for storage at ambient or refrigerated temperatures. The porouspolymer scaffolds to be used for tissue engineering and tissue guidedregeneration must also be sterile. Sterility may be readily accomplishedby conventional methods such as irradiation or treatment with gases orheat.

The following non-limiting examples set forth hereinbelow illustratecertain aspects of the invention. All parts and percentages are byweight unless otherwise noted and all temperatures are in degreesCelsius. Dicarboxylic acids and all other reagents were purchased inpure form and were used as received. Solvents were of “HPLC grade.” Alldiphenolic monomers (e.g., the esters of desamino tyrosil-tyrosine) wereprepared according to the procedure provided in Example I of U.S. Pat.No. 5,099,060. Although this procedure refers specifically to DTH,monomers having esters other than the hexyl ester can be readilyprepared by the same basic procedure. The DPTS catalyst was prepared asdescribed by Moore, et al., Macromol., 23 (1), 65-70 (1990).

EXAMPLES

The following table defines the abbreviations adopted for the diphenols,poly(alkylene oxide) dicarboxylic acids and polyarylates illustrated bythe examples below: TABLE I Desaminotyrosyl-tyrosine ethyl ester DTEDesaminotyrosyl-tyrosine hexyl ester DTH Desaminotyrosyl-tyrosine octylester DTO Desaminotyrosyl-tyrosine dodecyl ester DTD Poly(ethyleneglycol)-1000-succinate PEG-1K-suc Poly(DTE-PEG-1000) DTE-PEG-1000-sucPoly(ethylene glycol) 600 PEG-600 Poly(DTE-PEG-600) DTE-PEG-600Poly(ethylene glycol)-400-succinate PEG-400-suc Poly(DTE-PEG-400-suc)DTE-PEG-400-suc

Example 1 Preparation of DTE-PEG-1000-suc

2.644 g (0.0074 mols) of DTE and 0.653 g (0.0022 mols) of DPTS wereweighed into a 125 mL round-bottom flask containing an egg-shapedmagnetic stirring bar. 8.927 g of PEG-1000-succinate was weighed into a50 mL Ehrlenmeyer flask. Using 26 mL methylene chloride, the PEGcompound was transferred quantitatively to the round-bottom flask. Themixture was stirred until a pale yellow solution was obtained. 3.59 mLDIPC was added with stirring. The reaction mixture heated up and startedto boil. Within 20 to 30 minutes, the solution became very viscous andstirring stopped. The reaction was stirred overnight.

The polymer solution was added to 800 mL of isopropyl alcohol (IPA) withstirring. The polymer precipitated and collected as a ball. Afterdecanting the IPA solution, the polymer was shredded by hand and washedwith IPA. The polymer was dissolved in methylene chloride and washedwith water in a separatory funnel. The polymer was isolated from themethylene chloride solution by precipitation into IPA. The IPAprecipitate was further washed with two portions of ether and then driedunder high vacuum for 48 hours. Eight grams of a rubbery material with aweight-average molecular weight of 39,000 daltons was obtained.

Example 2 Preparation of DTE-PEG-600

Example 1 was repeated, substituting carboxymethyl PEG-600 forPEG-1000-succinate.

Example 3 Composites of PEG-Polyarylates with Calcium Sulfate

The polyarylate of Example 1 was mixed thoroughly with calcium sulfateparticles (particle size 10-20 mesh) with a stainless steel spatula. Thewt. % of calcium sulfate was varied from 10 to 95%. Approximately 2 g ofpolyarylate was mixed with each of the various weight percentages ofsolid calcium sulfate. The observations are summarized in Table II:.TABLE II Composites of DTE-PEG1K-suc with calcium sulfate (particle size10-20 mesh) Wt. % Calcium sulfate Observation 10 The polymer flows andtacky 25 The polymer flows and tacky 50 Polymer flows slower along withsolid particles. 75 Polymer flows relatively slower (than 50% composite)along with solid. Solid (calcium sulfate particles) and polymer settleat beaker bottom overnight 90 Composite can be shaped by hand; polymerdoesn't flow 95 Composite can be shaped by hand; polymer doesn't flow

Example 4 Drug Release Studies Using Injectable Polyarylates

Initial protein release studies were conducted using bovine serumalbumen (BSA) as the model protein. While BSA has no known therapeuticvalue, it was selected for several reasons. First, there is a largevolume of literature which allows a measure of success. Second, unliketherapeutic proteins and polypeptides, it is commercially available andinexpensive. Third, it is very stable. Finally, reliable analyticalmethods are available.

BSA was obtained from SIGMA (Cat # A 7906, Lot # 29H1282, Fraction V). Abuffer solution, 0.1M, pH 7.4 PBS was prepared by diluting PBS powderobtained from SIGMA (CAT # P3813) per directions supplied.

Acetonitrile (HPLC grade) was obtained from FISHBER (Cat #A998-4)Deionized water was collected from a BARNSTEAD Still. TFA was obtainedfrom ACROS.

A HPLC method was developed for assaying BSA. The column used for BSAanalysis was purchased from Phenomenex (Torrance, Calif.) with thefollowing specifications—Jupitor Stainless steel 250×4.60 mm, Silica C4fictionalized, 5-micron particle size, 300 A pore size. BSA was assayedby UV at 220 nm. The HPLC system used was a Perkin Elmer, equipped witha pump, UV detector and a Series 200 Auto sampler. Data was analyzedusing TURBOCHROM Version 6.0 Software.

Details of the BPLC method are summarized in Table III: TABLE III %Water (Distilled Deionized) % Acetonitrile Step Time, (TFA 0.1%) (TFA0.08%) 0 5 90 10 1 1 60 40 2 3 48 52 3 10 48 52 4 3 90 10

Under these conditions, BSA elutes as a sharp peak with a retention timearound 9.2 minutes (9.03 to 9.35). A calibration curve was constructedby sequential dilution of a 250 mg/25 mL (10 mg/ml) stock solution ofBSA in PBS. The concentration range was 0.4 mg/mL to 40-mg/mL. The curvewas found to be linear in this range, with a correlation coefficient of0.9918.

The robustness of BSA analysis was confirmed by checking thereproducibility of multiple injections. Two BSA samples (correspondingto the highest and lowest concentrations in the standard plot) wereprepared by dilution of a stock solution of BSA in PBS (BSA 0.2565 g in25 ml PBS) and injected 3 times in the HPLC. The retention times andArea (UV*sec) were found to be reproducible with a variation ofapproximately 5%. The detection limit for BSA under the methodologycurrently used was found to be 0.5 g/20 L. (0.025 mg/mL). A very smallpeak that cannot be quantitated was obtained at this concentration.

The quantification limit was determined by injecting sequentiallydiluted samples and estimating the amount of BSA. The results were thenconfirmed in a blinded study. The limit was defined as the concentrationat which BSA could be determined with an accuracy of 10%. This is arelatively large margin, which is acceptable for the initial screeningstudy, which will be narrowed during the development stage. Thequantification limit was established to be 1 mg/mL. Three concentrationsof BSA in PBS (n=3 for each) were provided blinded and the assayperformed. The operator was able to accurately identify and quantify thesamples within an error of around 10%. The standard deviation was verylow (<0.2 for all sets), suggesting very good accuracy. The peakretention times were between 9.09 and 9.12 minutes for all the unknownsamples, suggesting good reproducibility in terms of peak retentiontime. The peaks in all cases were a little asymmetric (distorted orbroad on right side). The analysis was more accurate in the lowerconcentration ranges. This was expected since the deviation from thecalibration curve is larger at the higher concentrations.

Preparation of DTE-PEG-400-suc

Example 1 was repeated, substituting PEG-400-suc for PEG-1000-succinate.

Formulation Methods

Standard Method. 500 mg of polymer is transferred into a 20 mLscintillation vial. 100 mg of BSA powder is added and stirred with aspatula until a homogeneous dispersion is obtained (about one minute,depending on polymer viscosity).

Formulation—Water Based. 500 mg of polymer is transferred into a 20 mLscintillation vial. An appropriate amount of water is added (0.5 to 2mL) and stir with a spatula until a homogeneous dispersion is obtained(again about one minute for low viscosity polymers). If the polymer MWis high, it may be necessary to place the mixture at overnight at 37° C.100 mg of BSA powder is added to the homogeneous dispersion and stirreduntil all the BSA is dissolved or dispersed.

This dispersion may be used as such or it can be freeze dried or driedunder vacuum. Attempts at freeze-drying have so far resulted in phaseseparation of BSA from the polymer. Formulations made by thismethodology have not been used in any release studies to date.

Formulation—Using Mini Max Extruder. One to two grams of polymer isplaced in the extruder and the extruder temperature was set at 35° C.The polymer is stirred at maximum speed for 30 minutes. After thetemperature stabilizes at 45° C. heating is stopped. 100 to 300 mg ofBSA is then mixed with the polymer. When the temperature drops to 35° C.the heating is turned on again for two minutes and then switched off.The BSA-polymer mixture is then stirred for about 6 minutes. The stirreris raised and lowered every minute. Finally the mixture is extruded intoa Teflon dish. The extruded mass is a ductile, opaque yellow solid, andcan be shaped by hand. The extruded mass can then be stored in afreezer.

Sample Preparation

0.1 g of BSA was dispersed in 0.5 g of polyarylate as described in thestandard formulation method section. 5 mL of acetonitrile (containing0.08% TFA) was added and the mixture was shaken overnight. To this, 45mL of PBS was added with shaking. Polymer precipitated and a clearsupernatant solution was obtained. The amount of BSA was quantified inthe supernatant by HPLC as described above.

In all formulations where BSA was directly mixed into the polymer, noseparate estimation of BSA was made and the loading was assumed to bethe same as the amount weighed. For two the melt-extruded formulations,BSA was estimated using the method described above. The extrusionprocess gives uniform distribution of BSA within the polymer matrix(error <5%).

Release Study

15 mL of PBS was added to the polymer-BSA formulation prepared asdescribed above. The vial was capped tightly and place in a 37° C.incubator shaker (rpm=200). At periodic intervals, 10 mL buffer wasremoved for analysis and replaced with 10 mL fresh buffer. For eachformulation, three replicates were used. The amount released wasreported as the average of the three replications. The BSA released wasquantified using the HPLC method described above.

The sampling intervals were 1 h, 3 h, 6 h, 12 h. 24 h, and 48 h. After24 h, the sampling interval can be reduced to once every 24 h or lessdepending on the rate of release.

BSA Release from DTR-PEG 600 Polymers

The release of BSA from DTR-PEG-600 (R₂=various desaminotyrosyl-tyrosineesters) injectable polyarylates was investigated. The aim of this studywas to investigate whether BSA can be released from these polymers andto study the effect of pendant chain on the release rates. The threepolymers used in this study had R₂=ethyl, hexyl and octyl pendantchains. The loading was 16.6%. Results that are averages of n=3 areshown in FIG. 1.

BSA was released from all polymers by a diffusion type process. Thefastest release was from the most hydrophilic polymer, DTE PEG 600,where the release was completed within 18 hours. The DTH polymer gavethe slowest release, which was complete within 48 hours. Interestingly,the release from the polymer with the most hydrophobic DTR moiety (DTO)was almost identical to that from the polymer with the most hydrophilicmoiety (DTE), and faster than the one with intermediate hydrophobicity(DTH). It is possible that the polymer properties are dominated by theproperties of PEG, which masks the influence of the pendant groups.

BSA Release from DTR-PEG 1000-Suc

Effect of DTR side Chain. These polymers differed structurally from theDTR PEG 600 polymers in that the PEG units are linked to the DTR unitsvia a succinic acid unit, whereas in the DTE PEG 600 polymers the DTRand PEG units are directly linked. Four polymers, containing DTE, DTH,DTO and DTD were used. The polymers were designed to have low Molecularweights (<15,000 KD) so that they could be formulated for injection,without added excipients or heating. The loading was 16.7% and therelease studies were conducted as described previously. The releasecurves are shown in FIG. 2.

All polymers released BSA in a diffusion type process. The rate ofrelease was related to the nature of the side chain on the DTR monomer,in the case of the DTE, DTH and DTO polymers. The release was completedwithin 18 hours for the polymer with the most hydrophilic side chain(DTE) while the polymer with the most hydrophobic chain (DTO), releasedBSA over a period of 96 hours and the one with intermediate length (DTH)released BSA over a 48 hour period. Interestingly, the polymer with themost hydrophobic chain (DTD), (which was expected to release BSA at theslowest rate), actually released BSA faster, with the release beingcompleted within 48 hours.

Influence of MW of PEG

DTE-PEG Suc with PEG molecular weights of 400 and 1000 were used. Thepolymers were rubbery materials and BSA could not be incorporated by theconventional method. The melt extrusion method was used and the loadingsestimated as discussed in previous sections. The loading for the DTE PEG400 succinate was 16.7% and for DTE-PEG 1000 Suc, it was 20%.

As shown in FIG. 3, the release from the lower molecular weight PEG 400polymer was slower than that from the PEG 1000 version. This is expectedsince the PEG 400 is less hydrophilic than PEG1000.60% BSA was releasedin 168 hours (7 days) from the PEG 400 polymer, while 80% BSA wasreleased in 5 days (120 hours) from the PEG 1000 polymer.

Effect of Polymer MW

DTE-PEG 1000-Suc with MW 35,000 and 12,000 was used (FIG. 4). Themolecular weight of the polymer had a very significant effect on therelease of BSA. While the release of BSA was complete in less than 1 daywith the LMW polymer, only 80% was released after 5 days with the HMWpolymer and the release continued for beyond 2 weeks, although at a veryslow rate.

Effect of Changing the PEG Structure: DTR-PLURONIC-Suc

In this family of polyarylate, the PEG unit is replaced with a morehydrophobic PLURONIC unit. DTE-PLURONIC-suc was synthesized as a typicalrepresentative of this family. BSA was incorporated into this materialat 16.7% loading by direct mixing and release studies initiated asbefore. The release curve obtained is shown in FIG. 5. After an initialburst of 10%, an additional 45% is released within 48 hours, after whichthe release seems to stop. A review of the BPLC traces showed that theBSA peak is broad and asymmetrical.

Polyarylate Degradation

The degradation of the polyarylates was monitored by GPC. (THF,polystyrene standards). Since the release rates are completed withinshort periods of time, the MW was measured at time zero, aftercompletion of release (about 1 week) and then at weekly intervals untilthe polymer is fully resorbed. The data for the are presented in thefollowing two tables (Tables IV and V). TABLE IV Degradation ofDTR-PEG600) ® = E, H, O) polymers Initial After Day 7 After Day 14 (N= 1) (N = 3) (N = 3) Polymer (M_(w)) (M_(w)) (M_(w)) DTE PEG 600 123008200(Shows another 4200 (Clearly shows two low MW peak and peaks. (Onelow mol. wt shoulder) peak) DTH PEG600 12600 2700 (Clearly shows twopeaks. (One low mol. wt peak) DTO PEG600 6800 2900 (Clearly shows twopeaks. (One low mol. wt peak)

TABLE V Degradation of DTR-PEG1000-Suc ® = E, H, O, D) Before After Day7 After Day 14 Polymer (M_(w)) (M_(w)) (M_(w)) DTE-PEG 1000-Suc/LMW*16900 14700 13600 DTH-PEG 1000-Suc/LMW* 18800 16400 14600 DTO-PEG1000-Suc/LMW* 20050 15800 13800 DTD-PEG 1000-Suc/LMW* 16300 16300 14600

The polyarylates made from the PEG dicarboxylic acid degrade at a fasterrate than those made from PEG-Succinates. It is expected that these allpolymers will break down to monomeric units within 4 to 6 weeks. This isof significance since it will allow for repeated dosing of the samepatient without accumulation of polymer.

The foregoing examples and description of the preferred embodimentsshould be taken as illustrating, rather than as limiting the presentinvention as defined by the claims. As will be readily appreciated,numerous variations and combinations of the features set forth above canbe utilized without departing from the present invention as set forth inthe claims. Such variations are not regarded as a departure from thespirit and script of the invention, and all such variations are intendedto be included within the scope of the following claims.

1. A polyarylate comprising repeating units having the structure:

wherein R₁ is CH═CH or (CH₂)_(n) wherein n is from 0 to 18, inclusive;R₂ is selected from the group consisting of hydrogen and straight andbranched alkyl and alkylaryl groups containing up to 18 carbon atoms;and R has the structure selected from the group consisting of:—(CH₂—)_(a)O—[(CH₂—)_(a)CHR₃—O—]_(m)(CH₂—)_(a) and—R₄—C(═O)—O[(—CH₂)_(a)—CHR₃—O—]_(m)C(═O)—R₄— wherein a is from 1 to 3,inclusive, m is from 1 to 500,000, inclusive, R₃ is hydrogen or a loweralkyl group containing from one to four carbon atoms, and R₄ is selectedfrom the group consisting of a bond and straight and branched alkyl andalkylaryl groups containing up to 18 carbon atoms.
 2. The polyarylate ofclaim 1, wherein R₁ is —CH₂—CH₂—.
 3. The polyarylate of claim 1, whereinR₂ is selected from the group consisting of ethyl, butyl, hexyl, octyland benzyl groups.
 4. The polyarylate of claim 1, wherein a is 1, R₃ ishydrogen and m is between about 10 and about
 100. 5. The polyarylate ofclaim 1, wherein R₄ contains up to 12 carbon atoms.
 6. The polyarylateof claim 5, wherein R₄ is selected from the group consisting of—CH₂—CH₂—C(═O)—, —CH═CH—, —CH₂—CH(—OH)—, —CH₂—C(═O)— and (—CH₂—)_(z),wherein z is between 0 and 12, inclusive.
 7. The polyarylate of claim 1,in combination with hydroxyapatite, calcium sulfate, tricalciumphosphate, demineralized bone matrix or bioglass.
 8. An implantablemedical device comprising the polyarylate of claim
 1. 9. The implantablemedical device of claim 8, wherein said device is a stent, film orpolymeric scaffold for tissue engineering.
 10. The implantable medicaldevice of claim 8, wherein the surface of said device is coated withsaid polyarylate.
 11. The implantable medical device of claim 10,wherein said device is a stent, film or polymeric scaffold for tissueengineering.
 12. The implantable medical device of claim 8, comprising abiologically or pharmaceutically active compound in combination withsaid polyarylate, wherein said active compound is present in an amountsufficient for therapeutically effective site-specific or systemic drugdelivery.
 13. The implantable medical device of claim 12, wherein saidactive compound is a pharmacologically active protein, peptide, vaccineor gene.
 14. The implantable medical device of claim 12, wherein saidactive compound is covalently bonded to said polyarylate.
 15. Acontrolled drug delivery system comprising a biologically orpharmaceutically active compound in combination with the polyarylate ofclaim 1, wherein said active compound is present in an amount sufficientfor therapeutically effective site-specific or systemic drug delivery.16. The controlled drug delivery system of claim 15, wherein said activecompound is physically coated with said polyarylate.
 17. The controlleddrug delivery system of claim 15, wherein said active compound isphysically admixed with or covalently bonded to said polyarylate. 18.The controlled drug delivery system of claim 15, wherein said activecompound is embedded or dispersed in said polyarylate.
 19. Thecontrolled drug delivery system of claim 15, wherein said activecompound is a pharmacologically active protein, peptide, vaccine orgene.
 20. The controlled drug delivery system of claim 15, comprisingmicroparticles loaded with said active compound suspended in a matrix ofsaid polyarylate.
 21. The controlled drug delivery system of claim 20,wherein said active compound is a pharmacologically active protein,peptide, vaccine or gene.
 22. A method for site-specific or systemicdrug delivery comprising implanting in the body of a patient in needthereof the implantable medical device of claim 12, 13 or
 14. 23. Amethod for site-specific or systemic drug delivery comprisingadministering to a patient in need thereof the drug delivery systemclaim 15, 16, 17, 18, 19, 20 or
 21. 24. An implantable medical device inthe form of a sheet for use as a barrier for surgical adhesionprevention consisting essentially of the polyarylate of claim
 1. 25. Amethod for preventing the formation of adhesions between injured tissuescomprising inserting as a barrier between said injured tissues the sheetof claim
 30. 26. An injectable drug delivery system consisting of thepolyarylate of claim 1 and a biologically or pharmaceutically activecompound in an amount sufficient for therapeutically effectivesite-specific or systemic drug delivery.
 27. The injectable drugdelivery system of claim 26, wherein said active compound is apharmacologically active protein, peptide, vaccine or gene.
 28. Theinjectable drug delivery system of claim 26 or 27, whereinmicroparticles containing said active compound are suspended in saidpolyarylate.
 29. The injectable drug delivery system of claim 26, 27 or28, wherein said system is excipient-free.
 30. A method of forming anexcipient-free injectable drug delivery system comprising directlymixing the polyarylate of claim 1 and a biologically or pharmaceuticallyactive compound in an amount sufficient for therapeutically effectivesite-specific or systemic drug delivery.
 31. The method of claim 30,wherein microparticles comprising said active compound are mixed withsaid polyarylate.
 32. The method of claim 30 or 31, wherein said activecompound is a pharmacologically active protein, peptide, vaccine orgene.
 33. In a surgical method for repairing diseased or damaged bonetissue comprising applying to said tissue a putty-like substance, theimprovement comprising said putty-like substance comprising thepolyarylate of claim 1 or
 7. 34. In a cosmetic reconstructive surgerymethod comprising applying a filler material to soft tissues, theimprovement comprising said filler material consisting essentially ofthe polyarylate of claim
 1. 35. The method of claim 34, wherein saidfiller material consists of said polyarylate.
 36. The method of claim 34or 35, wherein said polyarylate is radio-opaque.
 37. A method ofregulating cellular attachment, migration and proliferation on apolymeric substrate, comprising contacting living cells, tissues orbiological fluids containing living cells with the polyarylate ofclaim
 1. 38. The method of claim 37, wherein said polyarylate is in theform of a coating on the surface of a medical implant.
 39. The method ofclaim 37, wherein said polyarylate is in the form of a film.
 40. Themethod of claim 37 wherein said polyarylate is in the form of apolymeric tissue scaffold.
 41. The method of claim 33 wherein saidpolyarylate is radio-opaque.